The present invention relates to measurements of spontaneous and stimulated cell activity and, more particularly, to a method, device and system employing a nano and micro integrated chip for positioning cells and determining cellular electrical and chemical activity, via electrical and/or optical signals.
Improved understanding of complex cell function is important in the fields of science and medicine. One of the key objectives in studies of networks of biochemical reactions is to uncover fundamental principles that govern the cell functioning and the structure and evolution of biological modules. For example, in the discipline of brain research, much effort is devoted to unraveling the rules of formation, function and degeneration of neural networks, which are the linking bridge from neurons to brain.
Many of the crucial studies that led to important understanding of cell function have been conducted in vivo in living animals. It is well recognized that the difficulties encountered using in vivo measurements (e.g., visualization, control of the chemical environment, parallel recording from many cells) are overcome via in vitro settings of tissue slices or cells grown in culture.
Over the past two decades, there has been a tremendous growth in experimental methods that allow for biochemical and biophysical investigations of single cells. Such methods include laser confocal microscopy imaging techniques that can be used to localize bioactive components in single cells and single organelles within cells [S. Maiti et al., Science, 275, 530-532, 1997], use of near field optical probes for pH measurements in the cell interior, and the like.
Knowledge of cell activity may also be gained by measuring and recording electrical potential changes occurring within a cell, which changes depend on the type of cells, age of the culture and external conditions such as temperature or chemical environment. Thus, precisely controlling the physical and chemical environment of a cell under study significantly enhances the value of the research. Intracellular and extracellular electrical measurements have application in research studies of nerve cell bodies and tissue culture cells such as smooth muscle, cardiac, and skeletal muscle cells. Such measurements and suitable display of the results thereof are also useful for demonstrations in teaching laboratories.
There are several major different technologies to measure the electrical activity of cells. Known in the art are techniques which are commonly called “patch clamp recordings” [O. P. Hamill et al., Pfleugers Arch. 391, 85-100, 1981), which have developed into a very versatile and precise methods. These techniques allow researchers to observe the functioning of a single ionic channel, while monitoring neurons electrical activity in the brain, or allow the monitoring of the change in cell membrane area during a process of secretion, etc. The patch clamp technique provides exquisite resolution for measuring ionic currents in cell membranes, using a glass micropipette having an opening end of the order of 0.1 micron. The micropipette is filled with saline solution and is pressed gently onto the cell membrane, forming a stable physical high resistance electrical seal (in the GigaOhm range) on the cell membrane, commonly termed the Giga-seal. When suction is applied to the micropipette the cell membrane breaks and the cytoplasm and pipette solution start to intermix. Once this mixing is completed, the ionic environment in the cell is similar to the saline filling solution of the micropipette. Ionic currents in the cell membrane are thus indirectly determined by measuring the electrical potential of the solution filling the micropipette.
Another device for measuring the electrical activity of cells is an extracellular electrode, which is a microelectrode being attached to the cell membrane from the extracellular side. The capacitive coupling between the micro-electrode and the cell membrane alter the electrode potential which is used to determine and measure action potentials. As the extracellular electrode is only attached to the cell membrane from the outside, the cell membrane remains intact, and, provided that the appropriate conditions (temperature, PH etc.) are supplied to the cell culture, the cells can survive for weeks [R. Segev, M. Benveniste, Y. Shapira, E. Hulata, N. Cohen, E. Kapon and E. Ben-Jacob, “Long Term Behavior of Lithographically Prepared in vitro Neural Networks”, Phys. Rev. Lett., 88: 118102, 2001]. The extracellular signal is about a 1000 fold smaller than the intracellular signal and the noise level in the extracellular domain is of the order of 25 μV. On the other hand, the voltage of synaptic signals is typically lower than 2 μV. Hence, in the extracellular domain, synaptic signals exhibit a signal-to-noise ratio which is insufficient to allow detection of these signals by an extracellular electrode.
Also known in the art is an intracellular electrode entering the cell membrane to measure the intracellular voltage directly [B. Hille, “Ionic channels of excitable membranes”, SINAUER, Sunderland, Mass., 1992]. One such intracellular electrode is a fine wire with a sharpened point, where electrical signals which are detected by the sharpened electrode end are amplified, displayed or recorded by equipments electrically coupled to the electrode.
Intracellular electrodes are particularly useful in the elucidation of the single neuron dynamics. The main advantage of intracellular electrodes over the extracellular electrodes and the patch clamp is the high resolution measurements of the cellular voltage which allows studying the effect of a single synapse on a single neuron [H. Markram and M. Tsodyks, Nature, 382:807-810, 1996]. However, in this technology, during the measurements the cell membrane is damaged, causing the cellular organs and cytoplasm to diffuse out from the cell and as a result, within several hours (or less) the cell dies. Hence, the presently available intracellular electrodes cannot be used for long period experiments.
Recently, the study of electrical activity in cells has reached a turning point with the development of a multi-electrode-array (MEA) [Y. Jimbo et al., “Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture”, IEEE Trans. Biom. Egin., 40:804-810, 1993; B. C Wheeler and G. J. Brewer, “Multi-Neuron Patterning and Recording”, Enabling Technologies for Cultured Neuronal Networks, Editors D. A. Stenger and T. M. McKenna, Academic Press, page 167, 1994; G. J. A. Ramakers et al., “Culturing of Cerebral Cortex Neurons on Multi-Electrode Plates for the investigation of Long-Term Neuronal Network Development”, International meeting on substrate-integrated microelectrode arrays: technology and applications, Reutlingen, Germany, Jun. 23-26, 1998, abstract book page 21; M. Camepari et al., “Experimental Analysis of Neuronal Dynamics in Cultured Cortical Networks and Transitions Between Different Patterns of Activity”, Biol. Cyber., 77:153-162, 1997].
The MEA is an arrangement of 60 micro-electrodes which are used for parallel for recording the electrical activity of cells in a tissue slice or of cells grown in culture (such as a neural network). The electrodes, typically 10-30 μm in diameter and 50-500 μm apart, are connected to a processing unit via an arrangement of amplifiers. The MEA allows studying the effect of different chemicals or drugs on the electrical activity of the tissue slice or the cells in culture. While measuring the electrical activity via the MEA, other cell characteristics (e.g., the morphology of the cells, chemical activity of the cells, and the like) may be detected by other means, for example, light microscope, etc.
In a typical neural network experiment, a network is composed of about 106 neurons and glial cells, which are grown directly on top of the MEA. Neurons, which are loosely placed above a particular electrode of the MEA form capacitance coupling with that electrode, hence allowing monitoring and recording of both the electrical activity and the electrical stimulation of the neuron.
MEA is also useful in the area of drug discovery where the search for new compounds for clinical use is done by industrialized process. Typically, in a single drug discovery process, many chemical compounds are produced by chemists, where about one tenth of these compounds are qualified for chemical screening. The remaining compounds are further screened in a cell cultural screening, organ screening and animal screening, where in each screening about 9 tenth are disqualified for further research and only one tenth are qualified for further study. Statistically, of the remaining compounds (only about a dozen compounds are left after the above screening procedures) one third are found to achieve the desired clinical goal and only one tenth is approved by the Food and Drug Administration. Hence, the process of discovering a single drug which is eventually approved for clinical use begins with the screening of about one million compounds.
The main goal of each stage in drug discovery is, of course, to estimate the clinical effect of the compound on humans. Hence, experiments are being performed by applying the tested chemical on a model system, first an in vitro system (e.g., cell culture) and then an in vivo system (e.g., intact animals). It is appreciated that the in vivo tests are rather complicated and extremely expansive. Thus, the methodology of screening for new drug candidates demands new methodologies with which to implement efficient and high throughput screening at the early stages, where the compounds are tested in vitro.
Naturally, estimating the effect of a drug on a cell in culture is not an easy task. For example, even if one or more experiments are performed on a single cell, the conclusions from such experiment may not be applicable for a complex living animal or a human being. Thus, it is desired to conduct experiment on complex systems rather than on a single (or a few) cell. The MEA technology has been proposed to facilitate in drugs screening. Typically, however, only about 20 of the 60 electrodes of the MEA show electrical activity. Moreover, often the monitored activity cannot be attributed to a single cell, because more than one cells couples to a single electrode. Hence, although the MEA technology allows for measuring activity of many cells simultaneously, its ability to acquire knowledge on systems which are closer to the in vivo setup is limited.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method, system and device for determining cellular activity devoid of the above limitations.